Platelets possess specific, high-affinity, saturable receptors for factor XI, factor XIa, factor Xa, thrombin, high molecular weight kininogen, and factor Va and promote the proteolytic activation of factor XII, factor XI, factor X, and prothrombin. To investigate further the activated platelet surface as a locus for the molecular interactions of coagulation proteins, we have studied the binding of factor IXa and factor IX to platelets as well as the contribution of platelets to factor-X activation. Factor IX, isolated from human plasma by immunoaffinity purification utilizing a murine monoclonal antibody, was converted to factor IXa by incubation with purified factor XIa. We have demonstrated the specific, high-affinity, reversible binding of both factor IXa and factor IX to thrombin-activated platelets in the presence of calcium ions. Analysis of saturation binding data obtained under equilibrium conditions indicated the presence of 550 plus or minus 70 (mean plus or minus SD) sites per platelet for factor IXa with a dissociation constant of 2.5 plus or minus 0.5 nM, whereas there were 306 plus or minus 57 sites for factor IX with a Kd of 2.68 plus or minus 0.25 nM. Preliminary results suggest that platelet-bound factor IXa promotes half-maximal rates of factor-X activation at a concentration of 0.5 nM. The central hypothesis to be tested is that factor IXa is bound to high- affinity, specific platelet receptors with acceleration of factor-X activation as a functional consequence. Specifically, our objectives are to: 1) elucidate the biochemical basis and physiological significance of factor-IX and factor-IXa binding to platelets including the relationship between factor-IX and factor- IXa receptors; 2) determine the role (if any) of factor VIII and von Willebrand fator in binding of factor IX and factor IXa to platelets and for the platelet contribution to factor-X activation; 3) determine the state of platelet activation required for binding of factor IX and factor IXa to platelets and for the platelet contribution to factor-X activation; 4) elucidate the structural characteristics of factor IX and factor IXa required for binding to platelet receptors and for the platelet contribution to factor-X activation; 5) determine the functional consequences of factor- IXa binding to platelets by examining the kinetics of factor-X activation, utilizing chromogenic assays, proteolytic cleavage studies, coagulation assays and the release of an activation peptide from 3H-labeled factor X. These studies will provide essential information about the role of platelets in promoting the assembly of enzyme-cofactor-substrate complexes and the proteolytic activation of coagulation zymogens.